Membrane-mediated release of nucleotide from an initiator of chromosomal replication, Escherichia coli DnaA, occurs with insertion of a distinct region of the protein into the lipid bilayer.
DnaA protein, the initiator protein of E. coli chromosomal replication, can be rejuvenated from an inactive ADP form to active ATP-DnaA protein by acidic phospholipids in a fluid bilayer. Cross-linking studies with the photoactivable phospholipid analog 1-O-hexadecanoyl-2-O-[9-[[[2-[125I]iodo-4-(trifluoromethyl-3H- diazirin -3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine reveal insertion of DnaA protein into the hydrophobic region of the bilayer; this insertion is accompanied by membrane-mediated dissociation of the tightly bound allosteric nucleotides ADP and ATP. Photolabeling of DnaA protein occurred with membrane properties that resembled those needed for reactivation of ADP-DnaA protein; efficient labeling of DnaA protein was observed only when the lipid analog was incorporated into anionic vesicles and the temperature during treatment was above the gel to liquid crystalline phase transition. Predominant hydrophobic photolabeling was localized within a single region of DnaA protein, a region that contains putative amphipathic helices and has been shown to contain information essential for functional interaction with membranes.
Garner
J
J
Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, Washington, DC 20007, USA.
Durrer
P
P
Kitchen
J
J
Brunner
J
J
Crooke
E
E
D000227:Adenine Nucleotides / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000494:Allosteric Regulation; D001426:Bacterial Proteins / Q000378:metabolism*; D003432:Cross-Linking Reagents; D004261:DNA Replication; D004269:DNA, Bacterial / Q000096:biosynthesis; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli; D008051:Lipid Bilayers / Q000378:metabolism*; D008560:Membrane Fluidity; D010449:Peptide Mapping; D010743:Phospholipids / Q000378:metabolism*; D019920:Photoaffinity Labels; D011485:Protein Binding; D017433:Protein Structure, Secondary
D000227:Adenine Nucleotides; D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D008051:Lipid Bilayers; D010743:Phospholipids; D019920:Photoaffinity Labels; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate